Figure 1.
The chemical structure of delphinidin-3-glucoside (Dp).
Figure 2.
The schematic depicts the overall design of the animal experiment to evaluate the Dp content in the blood and blood vessels of BABL/c mice.
Figure 3.
Dp attenuates oxLDL-induced injury in HUVECs.
Confluent HUVECs were pretreated for 2 h with various Dp doses (0.1, 1, 10, 50, 100, and 200 μM, respectively). After the supernatant was removed, the cells were exposed to 100μg/mL of oxLDL for 12 h. (A) Cell viability was determined by using MTT assay and data are expressed as the percentage of the control (CTL). There was a significant deterioration in the cell viability parameters after oxLDL treatment compared to CTL (##P<0.01) and Dp pretreatment dose-dependently protected against the oxLDL-induced alterations. High Dp concentration ameliorated cell viability (**P<0.05). (B) Representative images of flow cytometric analysis by annexin-V-FITC/PI dual staining. The right bottom quadrant represents the Annexin-V-stained cells (early-phase apoptotic cells) and the top right quadrant represents PI- and Annexin V-stained cells (late-phase apoptotic/necrotic cells). (C) Apoptotic cells represent the percentage of Annexin V single positive and Annexin V/PI double positive cells (a-h). All results are presented as mean ± SEM. for at least three independent experiments. ##P<0.01,vs. the control group; *P<0.05,**P<0.01 vs. oxLDL-treated group.
Figure 4.
Effects of Dp on mitochondrial dysfunction in oxLDL-induced HUVECs.
HUVECs were pretreated with various Dp concentrations (1, 10, 50 and 100 μM, respectively) for 2 h, and then exposed to 100 μg/mL of oxLDL for another 24 h. (A) Intracellular ROS levels were estimated using the probe DCFH-DA. Fluorescence was read at 485 nm for excitation and 520 nm for emission. (B) Intracellular O2·− levels were estimated using the probe DHE. Fluorescence was read at 355 nm for excitation and 420 nm for emission. (C) Determination of ΔΨm was carried out using CLSM and fluorescences were determined by spectrophotometry assay. Red fluorescence is emitted by JC-1 aggregates in healthy mitochondria with polarized inner mitochondrial membranes, while green fluorescence was emitted by cytosolic JC-1 monomers and indicates ΔΨm dissipation. Merged images indicated the co-localization of JC-1 aggregates and monomers. The ΔΨm in each group was calculated as the ratio of red to green fluorescence. (D) ΔΨm was detected by flow cytometry assay. The designated R2 and R3 regions represent cell populations that exhibit high (R2) or low (R4) red-to-green fluorescence ratio, consistent with high and low Δψm, respectively. Results shown are one representative of three separate experiments and the proportion of cells in R3 regions were quantified and expressed as percent cells with decreased ΔΨm. (E) The mPTP opening was assayed using the calcein–cobalt quenching method. Different HUVEC groups were used to measure the normalized relative fluorescence units (NRFU) of calcein. Fluorescences were determined using an InfiniteTM M200 Microplate Reader (Tecan Group Ltd., Männedorf, Switzerland). All results are presented as means ± SEM. and images were representative of at least three independent experiments. ##P<0.05, versus the control group; *P<0.05, **P<0.01 versus oxLDL-treated group.
Figure 5.
Determination of Dp uptake by VECs.
(A) The autofluorescent emission spectra of Dp and ethanol (baseline). The peak emission wavelength was recorded at approximately 370 nm. (B) HUVECs were treated with 10 μM Dp for 2 h and the subcellular Dp distribution was measured using a CLSM (b, ×400 and d, ×1000 magnification) and with a contrast phase microscope (a, ×400 and c,×1000 magnification). (C) Evaluation of intracellular Dp content in HUVECs via HPLC. After incubation, cells were lysed and the supernatant was collected for HPLC analysis. The blank control cells was treated with 0.1% DMSO. The excitation spectra of the fluorescence for λem = 370 nm is presented in arbitrary units (a.u.). (D-E) Time, dos-, and temperature-dependent uptake of Dp in HUVECs was measured via HPLC. (D) HUVECs were treated with 10 μM Dp at 37 (▪) and 4°C (⧫) for the indicated times (10 min, 30 min, 1 h, 2 h, 4 h, and 24 h, respectively). (E) HUVECs were treated with different Dp concentrations (1, 10, 20, 50, and 100 μM, respectively) for 2 h at 37°C (▪) or 4°C (⧫), respectively. The difference between incorporated Dp by HUVECs at 37°C and that at 4°C represents the actively transported amount (▴). BABL/c mice were intravenously administrated different Dp doses or an equivalent volume of hydro-alcoholic solution. (F) Representative image showed the isolated thoracic aorta (red arrow). The Dp content in the blood (G) and isolated thoracic aorta (H) were measured by HPLC. The isolated thoracic aortas were separated, frozen sectioned, and then stained with PI to visualize the nuclei. (I) Representative fluorescent signals (white arrows) of Dp in blood vessels were observed by by CLSM (TCS SP2, Leica Microsystems GmbH). Values are presented as means ± SEM, n = 3.
Figure 6.
The role of SGLT1 in Dp uptake by HUVECs.
(A) Representative images showed SGLT1 expression on HUVECs and the isolated thoracic aorta of BABL/c mice by immuofluorescence assay. Red fluorescence is emitted by SGLT1 immunocomplexes (white arrows) and blue fluorescence is emitted by the nuclei stained with 4′,6′-diamidino-2-phenylindole (red arrows). (B-D) HUVECs were exposed to 10 μM Dp for 2 h at 37°C in the presence of normal sodium buffer (the control), sodium-free buffer, and culture medium supplemented with phlorizin or D-glucose, respectively. After 2 h of incubation, cells were washed and the intracellular Dp content was measured via HPLC. The effects of sodium (B), phlorizin (C), and D-glucose (D) on Dp uptake were detected, respectively. (E) SGLT1 was knocked-down by transfecting HUVECs with different doses of SGLT1 siRNA and the SGLT1 mRNA expression was determined by qRT-PCR. (F) The transfected cells were exposed to Dp (1, 10, 50, 100 and 200 µM, respectively) for 1 h at 37°C. Thereafter, the intracellular cntent of Dp per 106 cells was measured via HPLC. Values are presented as means ± SEM. (n = 3); ##P<0.01 vs. the control group.
Figure 7.
SGLT1 activity is essential for the inhibitive effects of Dp on mitochondrial dysfunction in HUVECs.
After the indicated treatements, the cells were harvested and lysed to detect protein levels of AIF, Cyt c, Bcl-2, Bax, Caspase-3, and β-Actin by western blot analysis. The blots are representative of three independent experiments and values are presented as means ± SEM. (n = 3); ## P<0.01 vs. the control group; *P<0.05, **P<0.01 vs. the oxLDL-treated group.
Figure 8.
Proposed pathways for Dp uptake by VECs and its inhibitive effects on mitochondrial dysfunction.